The objective of this research is to begin developing a method for total joint resurfacing with engineered cartilage. The approach is based on generating a biphasic construct of porous chitosan/calcium phosphate and hyaline cartilage, the cartilage having been produced by mesenchymal stem cells (MSCs) attached to and piled atop the scaffold. This proposal encompasses four aims: 1) Investigate the effects of cell seeding density and growth factor on the biochemical and mechanical properties of cartilage produced from human marrow-derived MSCs by the self-assembling process;2) Optimize integration and attachment of engineered cartilage to a porous chitosan-nanocrystalline calcium phosphate (chitosan/CaP) scaffold;3) Engineer a femoral head-sized hemispherical shell of cartilage attached to a mushroom-shaped chitosan/CaP scaffold;4) Use biphasic cartilage/CaP-chitosan constructs derived from allogenic bone marrow MSCs to repair critical size defects surgically created in the trochlear groove of New Zealand White rabbit femora. Specific Aim 1 deals just with enhancing chondrogenesis by MSCs in a scaffold-free model. Since MSCs are a scarce commodity it is important to understand how many will be needed for optimal chondrogenesis. We will also assess the effects of TGF-21, TGF-23, and BMP-13, alone and in combination, for accelerated chondroinduction and rapid accumulation of extracellular matrix. Specific Aim 2 is focused on bonding of the engineered cartilage to a rigid, porous, osteoconductive scaffold to which native bone could integrate to anchor the engineered cartilage. We will determine how scaffold microstructure and coating with fibronectin affect chondrogenesis and the cartilage-scaffold interfacial strength. Engineered cartilage produced as part of Specific Aims 1 and 2 will be evaluated based on histological, biochemical, and biomechanical properties. Specific Aim 3 adds the complexity of molding the engineered cartilage and supporting scaffold into a hemispherical shape as would be required for femoral head resurfacing. We will attempt to produce a uniform thickness layer of cartilage in a hemispherical shell attached to the porous chitosan/CaP scaffold. The thickness, sphericity, and coefficient of friction of this cartilage will be measured to assess its suitability for joint resurfacing. If successful, Specific Aim 4 will provide a proof-of-concept by restoring hyaline cartilage to a full-thickness defect in a living animal. This project will impact public health by demonstrating a feasible approach to joint resurfacing with engineered cartilage. This proposal targets a specific challenge, femoral head resurfacing, and represents the first steps necessary to overcome this challenge. PUBLIC HEALTH RELEVANCE: This project addresses important issues in the development of treatment alternatives for osteoarthritis, a degenerative disease that affects millions of Americans and is responsible for billions of dollars annually in lost wages and productivity. Cartilage tissue engineering has the potential for resurfacing an osteoarthritic joint with healthy, functional tissue so that joint replacement with metal, plastic, or ceramic components is unnecessary. This project develops a joint resurfacing strategy based on a biphasic construct of self-assembled cartilage attached to a porous calcium phosphate-chitosan composite scaffold.